Food allergies affect 15 million Americans and carry a high risk of life-threatening allergic reactions. Therefore, establishing its cause is critical to ong-term risk management. The production of allergen-specific IgE antibodies is a key mediator of these disorders. However, the B cell populations that are the source of IgE and how IgE production is controlled remain unclear. The overall goal of this project is to define the heterogeneity of human B cells that produce allergen-specific IgE using food allergy as a model. The basis for this project stems from initial observations by Platts-Mills and Commins that a novel form of food allergy related to IgE antibodies specific for galactose-?-1,3-galactose (alpha gal) results in delayed anaphylaxis in adult and pediatric patients after consumption of red meat. Alpha-gal is a blood group carbohydrate of nonprimate mammals and therefore is present in meat of animals that carry this carbohydrate. Remarkably, most of these patients had previously tolerated meat for years, suggesting that sensitization to alpha-gal occurred later in life leading to the production of alpha-gal specific IgE. The primary cause of these IgE antibodies is bites from ticks, which contain alpha-gal in the gastrointestinal tract. Thus, alpha-gal within ticks explains the relationship between tick exposure and sensitization to alpha-gal, with development of red meat allergy as a secondary event. In preliminary work, we assessed in vitro B cell responses to tick extract and found increased frequencies of memory B cells and plasma cells from PBMCs of allergic subjects but not healthy controls. We further observed greater activation and proliferation of class-switched B cells expressing markers previously associated with a memory phenotype. Based on these observations, we hypothesize that the alpha-gal IgE in patients with red meat allergy emerges from a memory B cell subset that responds to alpha-gal exposure via tick bites and after eating red meat. However, resolving the heterogeneity of peripheral B cells, including memory B cells, has been limited using standard cell analysis methods such as flow cytometry. We have applied mass cytometry by time-of-flight (CyTOF), which supports 40 markers in a single sample, for high- dimensional immune profiling of peripheral B cells from healthy donors and identified heterogeneous subsets within memory B cells that was consistent across individuals. This suggests that it may be possible to detect allergy-related changes in B cell populations and identify cellular sources of IgE in human peripheral blood samples using CyTOF. In this proposal we will build on these new findings to define the B cell compartment in red meat allergy subjects and determine the relationships between B cell populations and clinical data, and to determine the requirements for an alpha-gal specific IgE response following allergen exposure. Collectively, we expect these studies to yield new information for understanding the immune status of distinct B cell subsets in IgE-mediated food allergy, and better define immune signatures that inform global and allergen-specific B cell responses.